Journal of Immunological Methods, 151 (1992)237-244

237

© 1992Elsevier Science PublishersB.V. All righls reserved0022-1759/92/$05.00

JIM 06317

An immunochemical analysis of the human nuclear phosphoprotein p53 New monoclonal antibodies and epitope mapping using recombinant p53 B. Vojt6~ek ~, J. Bfirtek h, C.A. Midgley ~ and D.P. Lane ~ ~ Cancer Research Campa(~n Laboratories, Department of Biochemistry, Unicersity of Dundt'~; Dundee DDI 4HN, U~ and b Institute of tlaomatology and Blood Tt~nsfl~sion, Korunni 108. Prague I0, Czechoslovakia

IReceived25 November1991,revisedreceived 17 January 1992,accepted 10 F¢bruary 1992) Somatic mutation of the p53 gene is a very frequent event in the development of haman neoplasia, and germ line mutations in p53 are responsible for an inherited cancer susceptibility syndrome. Many of the mutations in p53 found in human tumours are point mutations that result in the substitution of a single amino acid in the protein. These point mutant proteins are much more stable than the normal protein and the mutant product accumulates to a high level which permits important information about p53 expre~Aon to be obtained by immunochemical analysis. Using bacterial egpression systems to produce fragments of human p53 we have isolated and characterised new monoclonal antibodies to p53. These antibodies are suitable for the measurement of p53 in ELISA, immunob[otting and immunoprecipitation analyses. They are especially useful in immunohistochemistry as they are able to react strongly with p53 in conventionally fLxed and processed histological sections. Key words." lramunochemicalanalysis;Nuclear pbosphoprotein p53; Moaoclonal aatibody; Epitope mapping

Introduction

The human p53 protein is a 393 amino acid nuclear phosphoprotein (reviewed in Lane and Benehimol, 1990; Levine etal., 1991). The normal protein has a very short half-life and thus is present in only minute amounts in normal tissues and cells. The protein is not essential for viability ~inee some turnout cells are completely unable to synthesize p53 due to inactivating mutations. However, the normal protein has a potent activity

Correspondence to: D.P. Lane, CRC Laboratories, University of Dundee, Dundee DDI 4HN, OK. Tel.: 0382-307920; Fax: 0382-24117.

as a negative regulator of cell growth. Restoration of normal p53 protein expression by transfection of the p53 gone will suppress the growth and tumorigenicity of many human cancer cells (Baker etal., 1990; Chen et al., 1990). Many tumour cells express high levels of p53 (Bartek etal., 1991) and in these cells molecular analysis suggests that the p53 protein is a mutant (Bartok etal., 1990; lggo etal., 1990; Rodrigues ctal., 1990; Bennett etal., 1991; Gusterson etal., 1991) although other mechanisms for the stabilisation of the normal protein in tumour cells may also be found (Reihsans etal., 1990; Oren et al., 1981). The accumulation of high levels of p53 is emerging as a potential novel marker for malignancy (Hall etal., 1991) and in certain tumour

238 types may be associated with poor prognosis (Cattoretti et al., 1988). This association may be explained by the growth-promoting activity associated with certain mutant p53 proteins since in transfection assays normal p53 behaves as a turnout suppressor but mutant p53 behaves as a dominant transforming oncogene (Finlay et al., 1989; Hinds et aL, 1989). To permit further investigation of p53 in human tumours we have raised new monoclonal antibodies (MAbs) to recombinant human p53. The new antibodies were selected for their ability to react with p53 in routine histological sections and three positive antibodies were extensively charactcrised to ensure their specificity and application to other techniques. The binding reactivities of these new antibodies Were compared with those of other antibodies to p53 by linear epitope mapping using a new deletion series of p53 expressed in bacteria and by steric cpitope mapping using a two site ELISA assay.

Materials and methods

Production of hybridomas Splenocytes from B A L B / c mice hyper-immunised with recombinant human wi~d-type p53 protein were fused with a non-producing mouse mycloma cell line X63AgS.653 using polyethylene glycol as a fusogen. Supernatants of selected hybrids were screened on fLxed monolayers of A431 and SVKI4 cell lines and Hsgl3T cell line by the indirect immunopcroxidase method. The specificity of MAbs DO-l, DO-2 and DO-7 for 053 was confirmed by immunoblotting, immnnoprocipitation and immunocytochemistry using a panel of cell lines with well-characterised homozygous p53 mutations (Bartek ctal., 1990; Rodrigues et al., 1990) as well as a series of tissue sections from both normal and tumour tissues fixed and processed in different ways (Bartek ct al., manuseript in preparation). Antibodies PAb 421 (Harlow et al., 1981), PAb 240 (Gannon et at., 1990), PAb 1005 (Thomas et al., 1983) and PAb 1801 (Banks et al., 1986) are all monoclonal antibodies that recognise human p53.

CM-1 is a rabbit polyclonal antibody raised against pure human p53 (Midgley et al., 1992), PAb 419 (Harlow et al., 1981) is a monoclonal antibody to SV40 large T antigen. The anti-vimentin antibody was obtained commcrciany (Dako Laboratories).

Cell culture All cells including human breast cancer cell lines (BT474, PMC42, BTS,t9, MDA231), human sarcoma cell lines RD and Hs913T, human vulval carcinoma cell line A431, coltm carcinoma line HT29, SV40-transformed keratinocyte ling SVKI4, monkey Cos cells, bovine BMGE cell line and mouse C6 and rat RK101 cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum (FBS). For some experiments, the exponentially growing cells were infected with SV40 virus at 37°C and cultured for a further 60 h before harvesting. Tissues and turnouts Tissue and turnout samples were obtained within 1-2 h of surgery, and either fixed for 15-20 h in methacarn (MC: a mixture of methanol, chloroform and acetic acid, 6 : 3 : 1 by volume), or routine 10% formol saline and embedded in paraffin, or snap frozen in liquid nitrogen. Immunohistochemical procedures Indirect immunoperoxidase assays were performed on either frozen tissue sections prefixed for 10 rain in a mixture of cold methanol and acetone (1 : 1 by volume) or deparaffiniscd sections. The mouse MAbs DO-l, DO-2 and DO-7 culture supernatant was diluted 1/10. The primary antibodies were detected by a sensitive streptavidin biotin peroxidase system (Vectastain Elite kit, Vector Laboratories, Burllngame, CA) employed as recommended by the manufacturer, with 3',3-diaminobenzidine in 0.03% nickel sulphate as the chromogen. The indirect immunoperoxidase and indirect immunofluorescence procedures were performed on tissue culture cells grown either on glass coverslips or on plastic dishes prefixed for 8 rain in a mixture of cold methanol and acetone (1:1 by volume). Cells were then incubated overnight at

239 4"C with monoelonal antibody supernatant diluted 1/10. The cells were then washed in PBS. Three changes of PBS were used for all washings between applications of the staining reagents. For indirect immunopernxidase staining procedures, peroxidase-eonjugated rabbit anti-monse immunoglobulin antiserum (Duke, Denmark) diluted 1/50 was used as the second antibody with 3',3-diaminobenzidine in 0.03% nickel sulphate as ehromogen. For indirect immonofluorescence assays the second antibody was an FITC-conjugated antiserum to mouse immonoglobulins (Duke, Denmark) diluted 1,/100 (Harlow and Lane, 1988).

Gel electrophoresis and immunoblotting For direct immunoblotting, the total cellular protein lysates were prepared by harvesting confluent cells in hot Laemmll ¢lectrophoresis sample buffer. Solubilised proteins were then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% gel and transferred onto a nitrocellulose membrane in a Bio-Rad Mini Trans-Blot Electrophoretic Transfer Cell for 4 h, at 4°(2 and 150 mA in transfer buffer (25 mM Tris, 190 mM glycine and 20% methanol) (Harlow and Lane, 1988). Prestained molecular weight markers (Biorad) were run in parallel. The blots were blocked in 0.1% Tween 20 in PBS for 1 h, probed overnight at 4°C with monoclonal antibody supernatant and washed in 0.1% Tween 20 in PBS. The blots were then incubated for 1 h at room temperature in Dako horseradish peroxidase (HRP)-eonjugated rabbit anti-mouse immunoglobulin a n t i , r u m diluted 1/100 and washed again in 0.1% Tween 20 in PBS. HRP was visualised with ehloronaphthol. lmmunoprecipitation Immunoprecipitation of the p53 protein from cells lysed in 150 mM NaCI, 50 mM "Iris, pH 8.0, 5 mM EDTA, 1% NP40, 1 mM PMSF with mouse MAbs was performed essentially as d > scribed previously (Gannon et al., 1990) using Protein G Sepbarose beads (Pbarmacia) for both pre-absorption of the lysates and isolation of the antibody-p53 complexes. After SDS-PAGE and blotting (see below), the immunopreeipitated p53 proteins were visualised

with rabbit anti-p53 antiserum (Gannon et al., 1990; Midgley et al., 1991) followed by pernxidase-conjugated swine antiserum to rabbit immunoglobulins (Dako, Copenhagen, Denmark) and ehloronaphthol (Sigma) as chromogen,

ELISA assay A sandwich immonoassay was used to assess the new MAbs DO-I, DO-2 and DO-7 in ELISA test performed essentially as described by Gannon et al. (1990) using munoclonal anti-p53 antibodies DO-I, DO-2 and !)O-7 as the solid-phase reagents and polyclonal rabbit antisera to p53 to detect the captured proteins. Falcon 96 well microtitre plates were incubated overnight at room temperature with 50 #.l/well of ascites fluid (diluted 1/500), rinsed in PBS and blocked for 2 h in 3% bovine serum albumin (BSA) in PBS. Cell extract was prepared as for immunoprecipitation; 50 ~1 of serial 2-fold dilutions in lysis buffer were added to each well and the plates incubated for 3 h at 4*C. The plates were washed in 0.1% NP40 in PBS, appropriate rabbit antiserum (diluted 1/1000) v,as added and the incubation continued for 3 b. The plates were then washed as above and peroxidase-conjugated swine antiserum to rabbit immuuoglobulin (DAKO, diluted 1/1000) was added for 2 h, visualised with tetrametbylbenzidine and the results monitored in an automatic ELISA plate reader (Harlow and Lane, 1988). 3' deletion series of human p53 Plasmid pT7-7 Hup53 which directs the expression of full-length p53 protein (Midgley et al., 1992) was digested with BamHI and Pstl. A series of uni-direetional deletions from the 3' end of the coding sequence were prepared using cxonuclease !II as described (Sambronk et aL, 1989). Recircularised plasmids were recovered and deletion end points determined by DI~IA sequencing. The truncated proteins were expressed by IPTG induction in the appropriate host. Results and discussion

The primary goal of the present fusion experiment was to produce MAbs which would be aP-

240 plicable for immunohistochemical detection of p53 in paraffin-embedded human tumour material. Mice immunised with the pure soluble recombinant human i)53 protein (Midgley et al., 1991) made a relatively good anti-p53 response as judged by initial cell-staining experiments. For the first screening hybridoma supernatants were tested in an immunopernxidase-staining assay on three cell lines: the A431 cell line which is known to express the mutant form of p53 protein, SVK14 which expresses high levels of normal p53 protein stabilised by the T antigen of virus SV40 and the Hs913T cell line which produces no p53 protein (Stratton et al., 1990). Out of several hundred hybridoma wells screened, fifteen gave clear positive nuclear staining on the A431 and SVK14 cells, but did not stain the nuclei of the Hs913T at all. After a subsequent immunoperoxidase screen on methacarn-fixed paraffin sections of a strongly p53 positive colon carcinoma three hybridomas were selected for further analysis. The cells were cloned once by limiting dilution and subsequently by manual single-cell cloning to ensure monoclonality. The immunoblotting analysis of SDSPAGE-separated whole cell lysates from several human cell lines known to express high levels of the I)53 protein revealed strong reactivity of these three monoclonal antibodies (DO-i, DO-2 and DO-7) with a 53 kDa band, which was also recog-

nised by control anti-p53 antibody (Fig. 1), while no detectable reaction of DO-I and DO-7 MAbs was seen on immunoblots of lysates from cell line Hs913T. Only MAb DO-2 recognised a 60 kDa band on p53 negative cell lines and 53 kDa and 60 kDa bands on p53 positive cell lines (Fig. 1). New bybridoma line DO-I secreted large amounts of an IgG2a antibody, DO-2 an IgG1 and DO-7 secreted large amounts of an IgG2b antibody. To further characterise the MAbs immunopredpitation and ELISA experiments on the different cell lines were performed as described in the materials and methods section. As can be seen from the data presented in Fig. 2, the control antibody and all three new MAbs immunoprecipitated human i)53 protein. MAb DO-2 did not precipitate the 60 kDa band and it is probable that this MAb recognises a denaturated epitope of the unknown 60 kDa protein. These MAbs were analysed in an ELISA test (Fig. 3). DO-I and DO-7 both acted as effective solid-phase reagents able to capture 1)53 from cell extracts. They were at least as effective as the previously characterised anti-p53 antibody PAb 421 in these assays. MAb DO-1 can be biotinylated (Biotinylation kit, Amersham International pie) and can then be used for detection of p53 as the labelled antibody using PAb 421 as the solid-phase capture antibody (Fig. 4). A deletion series of human p53 was used to

m

1

2

3

4

5

6

7

8

9

10

11

Fig. I. Characterizationof the monoclonalantibodiesDO-I, DO-2 and DO-7 by immunobloning.In immunoblotsof the whole-cell I'~satesof A431cells(lanes 1, 2, 3, 4, 5) the three new monoclonalantibodiesDO-I (lane l). DO-2(lane 2), DO.7 (lane 3) and the control PAb 42t (lane 4) showa positive53 kDa band whichis not seen usingthe negativecontrol antibodyPAb 419 (lane 5). On l-ls913Tcells(lanes6, 7. 8. 9,10, l 1)whichlackany p53 transcripts no reactivityis seen with MAb DO-t (lane 6), MAbDO-7 (lane 8). PAb 421 (lane 9) and negativecontrol antibodyPAb 419 (lane 10). Reactivityis seen onlywith positivecontrol anti-vimentin antibody(lane 1l) and anti-p53antibodyMAbDO-2(la[*e7) whichcross-reacts withan unknownproteinof 60 kDa.

241 1.280

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0.8'

i

0.6'

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0.4 0.2-

1

2

3

4

O.O . .01

5

Fig. 2. lmmunoprecipitation (IP) of p53 from cell line extract. Imntunoblot of immunoprccipitates from breast cancer cell line BT474. Extract was imntunoprecipitated with monoclonal antibodies DO.I (lane 1), DO-2 (lane 2), DO-7 (lane 3), PAb 421 (lane 4) and an irrelevant negative con,ol antibody PAb 419 (lane 5). Immunablote were probed with rabbit anti-p53 serum CM-I (indirect immunopemxidase detection method). Molecular weight of the maThers is given in kDa. characterisc the target epitopes of these M A b s (Fig. 5). All three antibodies bound to a fragment o f human p53 consisting o f the N terminal 45 amino acids (AA); they did not react with a fragment o f p53 consisting of the N terminal 37 amino acids. T h u s all three antibodies bind to the same linear a r e a o f p53 and the epitopes recognised are between amino acids 1 and 45 and

1.21.0-

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/

.

. .1

. . . . . 1 10 tOO Protein mgtml Fig. 4. Two site immunmmay using W1"4"/4cell extracl asd rabbit anti-p53 serum CM-I (0 4.) or biotin-labelled DO-I ([3 [32])antibody. The plates were coated with antibody PAb 421.

possibly between amino acids 37 and 45. All three antibodies not surprisingly compete sterically with each other for binding to i)53 in E L I S A procedures. However the t h r e e antibodies are clearly distinct clones because they a ~ of different subclasses. T h e precise epitope r e o ) ~ i s e d by D O - 2 is also distinguished from that recognised by D O - I and D O - 7 because only D O - 2 shows the cross-reaction with a 60 k D a protcil, in immunoblotting analysis. Since these epitopes potentially overlap in the linear sequence with that proposed to he recognised by P A b 1801 we analysed the binding

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Fig. 3. Two site immunoassay using BT474 cell extract to compare the reactivity of the new MAbs DO*I, DO-7 with PAb 421. The graph shows assays performed with MAbs DO-1 ( x x), DO-7 ( , A) and PAb 421 ([3 [3) as coating antibodies, probed with rabbit antip53 serum CM-1. Background levels were e~timated with the extract for the rabbit anti-p53 serum in the wells coated by a control antibody.

1 2 3 4 5 6 "/' Fig. 5.3' deletion series of human I)53. Direct immurmblots of SDS-PAGE-separatcd lysates from bacterial cells BL21(DF.3) plus containing only plasmld pTT-7(lane I), piasmid pT'/-7 with full length of human I)53 (lane 2), plasmid pT7.7 with 1-223 AA fragment of human p53 (lane 3), 1-205 AA flagment p53 (lane 4), 1-165 AA fragment p53 (lane 5), 1-45 AA fragment p53 (lane 6), 1-37 AA fragment of human p53 (lane 7).

242

a

b

C Fig. 6. Immunuhistochemical detection of p53 on frozen and paraffin sections of human breast carcinoma. (a) Frozen section stained with MAb DO-I; (b) paraffin section stained with MAb DO-I; (c) paraffin section slained with MAb DO-7.

o f t h i s antib~d~," to t h e d e l e t i o n m u t a n t s e r i e s . P A b 1801 d i d n o t r e a c t w i t h t h e 1 - 4 5 c o n s t r u c t but did bind to the 1-91 construct. Thus the epitopes rccognised by the DO series and PAb 1801 a r e d i s t i n c t in t h e l i n e a r s e q u e n c e o f p 5 3 .

T h e y a r e a l s o s t e r i c a l l y d i s t i n c t a s P A b 1801 w a s u n a b l e t o b l o c k t h e b i n d i n g o f D O - 1 t o p 5 3 in a n ELISA test. A d d i t i o n a l i m m u n o c y t o c h e m i c a l c h a r a c t e r i s a t i o n o n a p a n e l o f f i x e d cell l i n e s confirmed the reactivity of DO-l, DO-2 and DO-7

TABLE I MONOCLONAL ANTIBODIES RAISED AGAINST p53 PROTEIN AND THEIR BASIC CHARACTERISTICS Antibody

Target

Intensity of immunoslaining on

antigens

Cell

Frozen

Paraffin section

culture a

sections a

Melhacarn a

IP b

ELISA b

Binding ~ite fAA)

+ + + + + + +

+ + + + + +

1- 45 I - 45 1- 45 370-378 45-- 91 161-220 C terminus

Western 10% formalin

blot a

saline a DO-I DO-2 DO-7 421 1801 240 1005

p53 p53 + 60kDA p53 p53 p53 p53 p53

++ ++ + + + + + + + + ++

++ ++ + + + + + + + + + +

+++ +++ ++ + + -

+++ ++ + + + -

+++ +++ + + + + + + + + + + + +

Staining ntens y - = negative; + = weak positive; + + = positive; + + + = strongly positive, b Reactivity of immunoprecipitation tiP) and ELISA: + = reactivity; - = no reactivity.

243 with both wild-type and mutant human p53 and suggested that D O - l , D O - 2 and D O - 7 reeognise monkey and bovine p53, bat not mouse p53, as shown by positive staining of Cos cells, B M G E cells infected by SV40 virus and no staining of C6 and RK101 cell lines respectively. To eC~aluate their potential as a reagent for immunohistochemical analysis of aberrant p53 levels in tumour sections, a wide range of normal human tissues and carcinomas were immunostained with the D O - l , D O - 2 and D O - 7 antibodies using the sensitive biotin-streptavidin peroxidase procedure. All normal human tissues examined were negative including skin, brain, kidney, lung, stomach and breast. In contrast strong nuclear positivity confined to the a r e a of malignancy was seen in 60 different carcinoma cases including breast stomach and colon tumours previously shown by us (Bartok e t a l . , 1991 and unpublished observation) to express elevated levels of the p53 protein. in contrast 32 turnouts that did not show elevated levels of p53 in earlier work with polyelonal antip53 reagents were also negative with the new monoclonal antibodies. A comparative study o f different carcinomas pre-fLxed in different ways, as described in the materials and methods section, revealed that the M A b s D O - l , D O - 2 and D O - 7 can be used on frozen sections as well as methacarn or formol saline-fixed paraffin sections (Fig. 6 and Table l). T h e immunoperoxidase staining of D O - I , D O - 2 and D O - 7 was essentially identical to that obtained by the CM-1 polyclonal anti-p53 antiserum, the only reliable immunological reagent for detection of the p53 protein in paraffin-embedded material described to date. T h e results of the intmunoehemieal and immunohistological characterisation of the M A b s D O - l , D O - 2 and D O - 7 demonstrate their specificity for a denaturation-resistant epitope at the N terminus of p53 and suggest that they could be useful new tools for analysis of p53 expression in human mmours including immunohistochemistry on paraffin-embedded specimens.

Acknowledgements This research was supported by the Cancer Research Campaign. B,V. was supported by an

E M B O long-term fellowship; J.B. was supported in part by the U I C C Yamagiwa-Yoshida Memorial International Cancer Study Grant.

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244

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